FR3109153A1 - Compositions based on at least two glycosaminoglycans - Google Patents

Abstract

Compositions based on at least two glycosaminoglycans The present invention relates to a method of preparing supplemented glycosaminoglycan compositions which are flexible, stable, sterile and injectable. The compositions thus obtained comprise at least two types of glycosaminoglycanes, including at least one crosslinked glycosaminoglycan prepared from an uncrosslinked glycosaminoglycan of high molar mass and at least one uncrosslinked glycosaminoglycan of low molar mass. Figure for the abstract: None

Description

Compositions based on at least two glycosaminoglycans

The present invention relates to a method of preparing glycosaminoglycan supplemented compositions that are flexible, stable, sterile and injectable. The compositions thus obtained comprise at least two types of glycosaminoglycans, including at least one cross-linked glycosaminoglycan prepared from a non-cross-linked glycosaminoglycan of high molar mass and at least one non-cross-linked glycosaminoglycan of low molar mass.

Polysaccharides, such as glycosaminoglycans, are widely used in the biomedical field. In particular, the majority of products marketed for aesthetic and/or cosmetic applications are based on hyaluronic acid.

To improve the quality of the skin, unmodified hyaluronic acid gels are interesting because they have the advantage of being perfectly biocompatible, however, after implantation in vivo , they degrade very quickly. Thus, the effects of such gels are only short-lived.

In order to increase its durability in vivo and its resistance to degradation, hyaluronic acid is usually modified by cross-linking. Cross-linked hyaluronic acid gels can be obtained by different preparation methods. Polysaccharides, such as hyaluronic acid, can be cross-linked with different cross-linking agents, such as epoxy agents, aldehydes such as glutaraldehyde, divinyl sulfone (DVS) or polyamines. Currently, the agent most commonly used to crosslink hyaluronic acid is an epoxy agent, 1,4-butanediol diglycidyl ether (BDDE).

These crosslinking processes require two main steps, namely polysaccharide hydration and crosslinking.

The commercial products based on hyaluronic acid thus prepared generally comprise, and according to their indications, a fraction of cross-linked hyaluronic acid and a degree of modification ranging from 1% to 10%.

Nevertheless, increasing the cross-linking of hyaluronic acid leads to the preparation of a more chemically modified gel, therefore potentially less biocompatible. On the other hand, the mechanical performances of these compositions are limited and lead to a very elastic and/or brittle final product.

However, for reasons of product safety and performance, there is an interest in having durable compositions in vivo comprising a biocompatible polysaccharide, the most natural and the least modified, therefore the least crosslinked, possible.

In addition to the degree of cross-linking, the concentration of the glycosaminoglycan used is a key parameter in determining the mechanical and resistance properties of a composition. Thus, an alternative for increasing the in vivo durability and the resistance to degradation of a composition based on polysaccharides is to increase its total concentration in the final composition.

Nevertheless, in addition to increasing its resistance and its durability, the increase in the total concentration of said glycosaminoglycan leads to a thickening of the composition, with a significant increase in its viscosity. This increase in viscosity thus makes the gel difficult to inject and unable to integrate naturally into the tissues. In particular, the increase in the total concentration of hyaluronic acid is known to be the cause of the occurrence of post-injection papules.

Thus, for all these reasons, most products based on cross-linked hyaluronic acid marketed to date have a total hyaluronic acid concentration of only around 25 or 26 mg/g. However, although potentially useful for volumizing dermal filler applications, such gels are not suitable for cutaneous applications of regeneration and improvement of the appearance of the skin in which the mechanical filler is not the effect main target and which require injection into the superficial areas of the skin. For this last type of injection, it is in fact desirable to prepare a gel with lower elastic (G') and viscous (G'') moduli, which is flexible, easy to inject and which diffuses easily into the tissues.

Still in this context of increasing the resistance and persistence of products based on glycosaminoglycans, such as hyaluronic acid, it has also been proposed to add various compounds with protective properties against the acid. hyaluronic. Thus, against the degradation of hyaluronic acid by heat, application WO2014/032804 proposes the addition of magnesium ascorbyl phosphate, application WO2013/186493 that of sucrose octasulfate and application WO2007/077399 of glycerol. However, the addition of this type of stabilizing compounds increases the uncertainties regarding the biocompatibility of the products.

Consequently, there still remains a need for sterile gels based on glycosaminoglycans, and more particularly hyaluronic acid, which are flexible, easy to inject and which diffuse easily into the tissues, while being sufficiently durable and effective for regeneration and /or improving the appearance of the skin.

There remains, in particular, the need to increase the strength and durability of compositions based on glycosaminoglycans, such as hyaluronic acid, to overcome the problems of the prior art. Said invention proposes to meet these expectations.

According to a first aspect, the present invention relates to a method for preparing an aqueous gel comprising at least one cross-linked glycosaminoglycan and at least one non-cross-linked glycosaminoglycan, said method comprising at least the steps consisting in:

a) having an aqueous solution comprising at least one crosslinked glycosaminoglycan called "crosslinked HMM glycosaminoglycan" formed from uncrosslinked glycosaminoglycan with a molar mass greater than or equal to 0.5 MDa, preferably greater than 1 MDa, even more preferably comprised of 1 MDa to 4 MDa,

b) having an aqueous solution comprising at least one non-crosslinked glycosaminoglycan with a molar mass of from 0.04 MDa to 0.3 MDa, preferably from 0.08 MDa to 0.1 MDa; called “uncrosslinked FMM glycosaminoglycan” and,

c) forming a homogeneous mixture of all or part of said solutions of steps a) and b).

Against all expectations, the inventors have observed that the supplementation of compositions comprising at least one cross-linked glycosaminoglycan prepared from at least one non-cross-linked glycosaminoglycan of high molar mass with at least one non-cross-linked glycosaminoglycan of low molar mass amplifies the resistance of the final composition.

As can be seen from the examples below, it is observed:

better resistance to degradation compared to the same composition without added non-crosslinked low molecular weight glycosaminoglycan; And

better resistance to degradation compared to compositions having the same total concentration of glycosaminoglycan and the same degree of crosslinking of the crosslinked glycosaminoglycan fraction.

Because of this better resistance to degradation, the compositions according to the invention are more resistant to heat sterilization. Thus, between two sterile and injectable compositions of similar rheological properties before sterilization, one in accordance with the invention and the other prepared according to the teachings of the prior art, the first shows rheological properties after sterilization superior to those of the second.

Thanks to their increased resistance to degradation, the compositions according to the invention are advantageously capable of withstanding variable temperatures, in particular during their transport and their storage.

Furthermore, supplementation with at least one uncrosslinked glycosaminoglycan of low molar mass makes it possible to prepare compositions that are flexible, easy to inject and which diffuse easily with an increased total concentration of glycosaminoglycan, therefore with one or more biomechanical and/or biological effects on most important skin physiology(ies).

In addition, the use of a composition prepared according to the invention allows the simultaneous administration of glycosaminoglycans of different molar masses conducive to a diversification of the physiological effects of the glycosaminoglycan at the level of the treated site.

According to a second aspect, the present invention relates to a composition capable of being obtained according to a method in accordance with the invention.

The invention also relates to a composition according to the invention for its use in the prevention and/or treatment of an alteration in the viscoelastic or biomechanical properties of the skin.

It also relates to a composition according to the invention for its use in the prevention and/or treatment of cutaneous signs of chronological aging and/or induced by external factors such as stress, atmospheric pollution, tobacco or prolonged exposure. to ultraviolet (UV).

Preferably, the invention relates to a composition according to the invention for its use in the augmentation and/or the filling of soft tissues, in particular the filling of wrinkles.

Figure 1 is a graphical representation of the loss of elastic modulus (G') upon sterilization by autoclave (ΔG'(%)), the concentration of non-crosslinked low molecular weight hyaluronic acid and the concentration of hyaluronic acid cross-linked and non-cross-linked high molar mass hyaluronic acid from samples 1-1 to 1-3.

Figure 2 is a graph showing the losses of elastic modulus (G') upon sterilization by autoclave (ΔG'(%)) of sample 3-5 without added uncrosslinked hyaluronic acid and samples 3-6 to 3-9 comprising non-crosslinked hyaluronic acid added respectively of molar mass 0.04 MDa, 0.1 MDa, 0.2 MDa and 1.5 MDa.

Figure 3 is a graph showing the percentage improvements in loss of elastic modulus (G') on autoclave sterilization of Sample 3-5 without added uncrosslinked hyaluronic acid and Samples 3-6 with 3 -9 comprising non-crosslinked hyaluronic acid added respectively with a molar mass of 0.04 MDa, 0.1 MDa, 0.2 MDa and 1.5 MDa.

Figure 4 is a graphical representation of the loss of elastic modulus (G') upon sterilization by autoclave (ΔG'(%)), the concentration of uncrosslinked low molar mass hyaluronic acid and the concentration of hyaluronic acid cross-linked and non-cross-linked high molar mass hyaluronic acid from sample 4-1 (non-supplemented) and samples 4-2 to 4-4.

FIG. 5 is a graph showing the differences in elastic modulus (G′) before and after treatment with hyaluronidase (ΔG′(%)) of a sample 6-1 without added uncrosslinked hyaluronic acid and of samples 6-2 and 6-3 comprising non-crosslinked hyaluronic acid added with a molar mass of 0.1 MDa and 1.5 MDa respectively.

detailed description

Definition of terms according to the invention :

- THE "glycosaminoglycans » are defined as long chains composed of repeating disaccharide units of which one of the two carbohydrate residues is an amino carbohydrate (N-acetylglucosamine or N-acetylgalactosamine) and the second usually a uronic acid (glucuronic or iduronic). Glycosaminoglycans include, but are not limited to, hyaluronic acid, heparosan, chondroitin sulfate, dermatan sulfate, and keratan sulfate. A particularly preferred glycosaminoglycan is hyaluronic acid.

- By " hyaluronic acid », it is understood hyaluronic acid, or hyaluronan, and its derivatives. Therefore, the term “hyaluronic acid” also encompasses hyaluronic acid salts such as sodium hyaluronate and chemically modified hyaluronic acids, for example by oxidation, reduction, deacetylation, sulphation or amidation. Hyaluronic acid is a linear polysaccharide with alternating D-glucuronic acid and N-acetyl-D-glucosamine units, linked by alternating β-1,3 glycosidic and β-1,4 glycosidic bonds.

It is present naturally in several tissues of the human body and is degraded by enzymes, hyaluronidases, also present in the body, and/or via oxidation mechanisms.

- There" molar mass », expressed in g/mol or Da, of a glycosaminoglycan refers to the average molar mass of the glycosaminoglycan molecules used. The molar mass of a glycosaminoglycan can be determined according to various methods known to those skilled in the art, such as by capillary electrophoresis, by mass exclusion chromatography or even from a measurement of intrinsic viscosity.

- The " intrinsic viscosity ", ([η]), expressed in mL/g or m ³ /kg, of a glycosaminoglycan refers to the contribution of the glycosaminoglycan to the viscosity of a solution. It is measured by flow through a capillary viscometer. It represents the hydrodynamic specific volume of the polymer. The lower the intrinsic viscosity of a glycosaminoglycan, the lower its molar mass. For a given glycosaminoglycan, the intrinsic viscosity is linked to the molar mass by an empirical relationship, the Mark-Houwink relationship, which can be understood by any person skilled in the art, whose formula is as follows:

[η] = K x ^Mα

with :
K a constant depending on the polymer studied, the solvent used and the temperature, in particular, K is linked to the dimensions of the chains of the polymer studied;
α a constant depending on the polymer studied, the solvent used and the temperature, in particular, α is linked to the conformation of the polymer studied; And,
M the molecular molar mass of the polymer, expressed in Dalton (Da).

- By" glycosaminoglycan of low molar mass »say again " glycosaminoglycan F M M', it is understood a glycosaminoglycan with a molar mass ranging from 0.04 to 0.3 MDa, preferably from 0.08 MDa to 0.1 MDa, which corresponds to a glycosaminoglycan of low intrinsic viscosity, that is to say intrinsic viscosity ranging from 0.10 to 0.8 m³/kg preferably from 0.2 to 0.3 m³/kg measured according to the method indicated by the European Pharmacopoeia (edition 01/2017:0195).

- By" glycosaminoglycan from above e molar mass »say again " glycosaminoglycan H M M', it is understood a glycosaminoglycan with a molar mass greater than 0.5 MDa, preferably 1 MDa, more preferably between 1 MDa and 4 MDa, which corresponds to a glycosaminoglycan of high intrinsic viscosity, that is to say intrinsic viscosity greater than 0.8 m³/kg, preferably over 1.2 m³/ kg, more preferably comprised of 1.2 m³/kg at 3.5m³/kg measured according to the method indicated by the European Pharmacopoeia (edition 01/2017: 0195).

- A " glycosaminoglycan reticulated »means a glycosaminoglycan modified with a crosslinking agent during a crosslinking reaction. Conversely, a" glycosaminoglycan non cross-linked »means a glycosaminoglycan not modified with a crosslinking agent and which therefore has not undergone a crosslinking reaction.

The expression “ HMM glycosaminoglycan ” denotes the “crosslinked HMM glycosaminoglycan” component alone and the “uncrosslinked HMM glycosaminoglycan” component if present, in the aqueous gel.

- The " crosslinking rate " applied during the preparation of a "crosslinked glycosaminoglycan" corresponds to the ratio between the mass of crosslinking agent and the mass of glycosaminoglycan used for the preparation of said crosslinked glycosaminoglycan. It is expressed as a percentage.

- A cross-linked glycosaminoglycan can be qualified in particular by its "degree of change AVERAGE ». This degree of modification corresponds to the molar quantity of modifying agent bound to said glycosaminoglycan, by one or more of its ends, expressed per 100 disaccharide units of the glycosaminoglycan). It can be determined by methods known to those skilled in the art such as Nuclear Magnetic Resonance (NMR) spectroscopy.

- A" glycosaminoglycan reticle from above e molar mass (HMM) »or one" glycosaminoglycan from above e molar mass (HMM) reticulated »refers to a cross-linked glycosaminoglycan prepared from a non-cross-linked high molecular weight hyaluronic acid. Preferably, a crosslinked HMM glycosaminoglycan has a crosslinking rate of less than or equal to 8%, preferably less than or equal to 5%, and even more preferably less than 2%.

- " I are biomechanical and biological properties of glycosaminoglycan s on the physiology of the skin »include hydration and fibroblast activation. For example, due to its biomechanical and biological properties, hyaluronic acid participates in the restructuring, regeneration and/or rejuvenation of the skin.

- The " supplementation " of a composition represents the addition in this composition of at least one non-crosslinked glycosaminoglycan of low molar mass. Preferably, the supplementation corresponds to the addition in a composition of at least one non-crosslinked hyaluronic acid of low molar mass.

- By"total concentration of glycosaminoglycan », it is understood the sum of the concentration of cross-linked and/or non-cross-linked high molar mass glycosaminoglycan and the concentration of the glycosaminoglycan used for the supplementation.

- A solution is considered to be " homogeneous " when its color, visual appearance and viscosity are perceived, to the naked eye and to the touch, as uniform.

- By “ aqueous media ” is meant any liquid medium containing at least water as solvent and which has the property of dissolving a glycosaminoglycan.

- “ Crosslinking agent ” means any compound capable of introducing crosslinking between different glycosaminoglycan chains. By way of crosslinking agent suitable for the implementation of the present invention, mention may in particular be made of epoxydic or non-epoxidic bi- or multifunctional crosslinking agents. Among the epoxy agents, mention may be made of butanediol diglycidyl ether (BDDE), diepoxy-octane, 1,2-bis-(2,3-epoxypropyl)-2,3-ethylene, 1,2-bis(2,3 -epoxypropyl)-2,3-emylene and mixtures thereof. Among the non-epoxidic agents, mention may be made of endogenous polyamines such as spermine, spermidine and putrescine, aldehydes, carbodiimides and divinylsulfone.

- By " soft gel " is meant a gel whose viscoelastic distribution remains moderately elastic , i.e. sufficiently viscous with a phase angle (δ) of between 15° and 45°, a low elastic modulus G', in other words a G' less than or equal to 150 Pa, and an extrusion force less than or equal to 18N, preferably less than 15 N, measured at a fixed speed of approximately 12.5 mm/min, in syringes of which the internal diameter is greater than or equal to 6.3 mm, with a needle of external diameter less than or equal to 0.3 mm (30 G) and length ½ '', at room temperature.

- By “ easy to inject ” is meant a gel having an average extrusion force of less than 18 N, preferably less than 15 N, when measured with a dynamometer, at a fixed speed of approximately 12, 5 mm/min, in syringes of external diameter greater than or equal to 6.3 mm, with a needle of external diameter less than or equal to 0.3 mm (30 G) and length ½ '', at room temperature.

- By composition or gel " diffusing easily into the tissues " is meant a composition for cutaneous applications of regeneration and improvement of the appearance of the skin which does not promote the formation of papules after its injection into the superficial areas of the skin, ie in the upper dermis and the middle dermis.

- By" sustainability live » , it is understood the ability of the gel to remain at the treated site over time after its injection. A lasting gelliveis a gel capable of remaining at the treated site for at least 1 month, preferably at least 3 months, even more preferably at least 6 months, that is to say capable of maintaining after application an overall aesthetic improvement for at least 1 months, preferably at least 3 months more preferably at least 6 months, in other words capable of maintaining after application at least a grade 1 or 2 of improvement on a subjective scale for evaluating the overall aesthetic improvement comprising 5 grades: 1. Much improved; 2. Improved; 3. Unchanged; 4. Gradient; 5. Very degraded and this, for at least 1 month, preferentially at least 3 months, still preferentially at least 6 months.

- The term "stability"characterizes the ability of a composition to maintain physico-chemical properties (color, pH, homogeneity, turbidity, rheology, etc.) in accordance with its specifications, during storage for at least one year. The stability of a composition is generally monitored by observing and/or measuring one or more of its physico-chemical parameters, in particular one or more of its rheological parameters, such as its elastic modulus (G') or its viscosity ( η).

- By “ sterile gel ” is meant to qualify a gel endowed with the safety required for administration in or through the superficial zones of the skin, ie in the upper dermis and the middle dermis. In particular, it is essential that said gel to be administered using an injection technique be devoid of any contaminating body capable of initiating an undesirable side reaction in the host organism.

- The terms “additive ” , “additional component” and “excipient” are used interchangeably and designate any component compatible with an application in and/or on the skin. The amount of additive used depends on the nature of said component, on the desired effect, and on the use of the composition according to the invention. This additive may be one of the following components or one of its derivatives: an anesthetic agent, an antioxidant, an amino acid, a vitamin, minerals, a nucleic acid, a nucleotide, a nucleoside, a co-enzyme, an adrenergic derivative, sodium dihydrogen phosphate mono-hydrate and/or di-hydrate or sodium chloride.

- As " anesthetic agent ", there may be mentioned Ambucaine, Amoxecaine, Amyleine, Aprindine, Aptocaine, Articaine, Benzocaine, Betoxycaine, Bupivacaine, Butacaine, Butamben, Butanilicaine, Chlorobutanol, Chloroprocaine, Cinchocaine, Clodacaine, Cocaine, Cryofluorane, Cyclomethycaine, Dexivacaine, Diamocaine, Diperodon, Dyclonine, Etidocaine, Euprocine, Febuverine, Fomocaine, Guafecainol, Heptacaine, Hexylcaine, Hydroxyprocaine, Hydroxytetracaine, Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamidine, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine, Myrtecaine, Octacaine, Octodrine, Oxetacaine, Oxybuprocaine, Parethoxycaine, Paridocaine, Phenacaine, Piperocaine, Piridocaine, Polidocanol, Pramocaine, Prilocaine, Procaine, Propanocaine, Propipocaine, Propoxycaine, Proxymetacaine, Pyrrocaine, Quatacaine, Quinisocaine, Risocaine, Rodocaine, Ropivacaine, Tetracaine, Tolycaine, Trimecaine, and their salts.

- As " antioxidant " , it can be mentioned glutathione, ellagic acid, spermine, resveratrol, retinol, L-carnitine, polyols, polyphenols, flavonols, theaflavins, catechins, caffeine, ubiquinol, ubiquinone, alpha-lipoic acid.

- As " amino acids " may be mentioned arginine, isoleucine, leucine, lysine, glycine, valine, threonine, proline, methionine, histidine, phenylalanine, tryptophan, cysteine.

- As “ vitamin e s ” , there may be mentioned vitamins E, A, C, B, and in particular vitamins B4, B5, B6, B8, B9, B7, B12 and preferentially pyridoxine.

- As " minerals " , it can be mentioned the salts of zinc, magnesium, calcium, potassium, manganese, sodium, copper.

- As " co-enzymes " , it can be mentioned the coenzymes Q10, CoA, NAD, NADP.

- As " adrenergic derivative " , it can be mentioned adrenaline and noradrenaline.

Finally, it is understood that all the numerical values indicated are not to be considered strictly but approximately according to the general convention, in other words it is the last digit indicated of the numerical values which corresponds to the precision of the measurement. For example, for a molar mass of 0.04 MDa, the margin of error is 0.035-0.044 MDa.

METHOD ACCORDING TO THE INVENTION

As specified above, the present invention relates to a method for preparing an aqueous gel requiring, on the one hand, an aqueous solution comprising at least one crosslinked glycosaminoglycan, also called "crosslinked glycosaminoglycan HMM" because it is formed from non-crosslinked glycosaminoglycan with a molar mass greater than or equal to 0.5 MDa, and on the other hand, an aqueous solution comprising at least one non-crosslinked glycosaminoglycan with a molar mass of 0.04 MDa to 0.3 MDa, known as " non-crosslinked FMM glycosaminoglycan” and, to form a homogeneous mixture of all or part of said solutions.

Preferably, the glycosaminoglycans used in a process according to the invention are chosen from hyaluronic acid, heparosan, chondroitin sulphate, dermatan sulphate, keratan sulphate and mixtures thereof.

The glycosaminoglycans used in the method of the invention may be of identical or different chemical nature.

In a particular embodiment, the glycosaminoglycans used in the process of the invention are of identical chemical nature and preferably are all hyaluronic acid.

As indicated previously, the method according to the invention uses these glycosaminoglycans and preferably hyaluronic acids in the form of aqueous solutions in particular as defined in steps a) and b) of the method according to the invention.

These solutions of glycosaminoglycans are advantageously homogeneous.

They can be prepared according to a conventional method, for example by solubilization and/or dilution of a crosslinked or non-crosslinked glycosaminoglycan in an aqueous medium, such as saline phosphate buffer. These preparations can advantageously be carried out at room temperature, preferably at a temperature between 15° C. and 25° C., with mechanical or manual stirring.

STEP A)

As regards the aqueous solution of "crosslinked HMM glycosaminoglycan" considered in step a), it is obtained beforehand by crosslinking at least one non-crosslinked glycosaminoglycan with a molar mass greater than or equal to 0.5 MDa, preferably greater than 1 MDa, again preferentially comprised from 1 MDa to 4 MDa with at least one crosslinking agent.

The realization of such crosslinking is clearly within the competence of the person skilled in the art.

Advantageously, the crosslinking agent is introduced in a ratio between the mass of crosslinking agent and the mass of uncrosslinked glycosaminoglycan used for the preparation of said crosslinked glycosaminoglycan, i.e. at a crosslinking rate, less than or equal to 8% by mass, preferably less than or equal to 5%, more preferably still less than 2%, ie in a molar ratio relative to the total number of moles of glycosaminoglycan units comprised from 0.001 to 0.25.

This cross-linking can be carried out at various temperatures and times to obtain the expected degree of modification.

Thus, a crosslinking carried out at room temperature, that is to say a temperature varying from 15° C. to 25° C., may require a reaction time greater than 5 hours, even 10 hours, or even more than 10 hours.

On the other hand, crosslinking stimulated by a stimulating element such as heating, exposure to UV radiation, exposure to microwaves or a catalyst, preferably through the use of heating, can see its rate of crosslinking significantly increased. and its duration may then be less than 5 hours.

In a particular embodiment, the crosslinking of the non-crosslinked glycosaminoglycan with a molar mass greater than or equal to 0.5 MDa, preferably greater than 1 MDa, even more preferably comprised from 1 MDa to 4 MDa is carried out at a temperature greater than 40° C. , preferably greater than 50°C, more particularly comprised from 45°C to 60°C or better still, comprised from 50°C to 55°C.

Such crosslinking can advantageously have a duration of 30 to 300 minutes, preferably 100 to 240 minutes.

Preferably, the crosslinking agent used in a method according to the invention is an epoxy crosslinking agent, preferably 1,4-butanediol diglycidyl ether (BDDE).

In a particular embodiment where the crosslinking agent used is a bi- or multifunctional epoxy crosslinking agent, like BDDE, the pH of the aqueous medium is basified or acidified in order to activate the crosslinking reaction . According to a particular mode, the aqueous medium is basified with generally a solution containing sodium hydroxide. Preferably, the pH of the aqueous medium is greater than or equal to 11, even more preferably, the pH of the aqueous medium is greater than or equal to 12.

According to a preferred embodiment, the concentration of “crosslinked HMM glycosaminoglycan” of the aqueous medium from step a) is adjusted to form, at the end of step c), an aqueous gel whose concentration of “crosslinked HMM glycosaminoglycan” is at least 5 mg/g relative to the total weight of said aqueous gel.

STEP B)

Step b) of the process of the invention requires for its part to have an aqueous solution comprising at least one “uncrosslinked FMM glycosaminoglycan”.

Preferably, the concentration of "uncrosslinked FMM glycosaminoglycan" considered in the aqueous solution of step b) is adjusted to form at the end of step c) an aqueous gel whose "uncrosslinked FMM glycosaminoglycan" concentration is d at least 3 mg/g, preferably 10 mg/g relative to the total weight of said aqueous gel.

As detailed below, the process according to the invention can also comprise the implementation of a glycosaminoglycan distinct from those defined for steps a) and b).

STEP C)

As described previously, step c) consists in forming a homogeneous mixture from at least all or part of the solutions obtained in steps a) and b).

The formation of a homogeneous mixture as considered in step c) advantageously involves at least one homogenization step.

Homogenization can advantageously be carried out under mild conditions in order to prevent degradation of the chains of the glycosaminoglycans used.

This step c) of forming a homogeneous mixture of at least one cross-linked glycosaminoglycan and at least one non-cross-linked glycosaminoglycan can be carried out according to conventional homogenization methods, such as three-dimensional agitation, agitation by mixer with blades, agitation with a spatula and/or an extrusion step through at least one screen.

According to a preferred mode, step c) comprises at least one step of extruding the mixture through at least one grid. Adjusting the characteristics of such a grid falls within the general knowledge of those skilled in the art. Conventionally, they are chosen according to the properties expected for the mixture to be formed and taking into account the respective specificities of the solutions of glycosaminoglycans brought together.

According to a particular embodiment, this step c) can thus comprise at least one step of homogenization by means of a paddle mixer, followed by at least one step of extruding the mixture through at least one grid. .

Homogenization is considered satisfactory when the gel obtained presents macroscopically, to the naked eye and to the touch, a homogeneous coloration, a uniform viscosity and is devoid of agglomerates. The duration of the homogenization is assessed by those skilled in the art according to their general knowledge. Said homogenization may comprise several cycles, optionally spaced out in time by a rest phase, in particular so as to assess the quality of homogenization of the glycosaminoglycans within said gel.

More particularly, a step c) according to the present invention can comprise homogenization for a total duration of less than 200 minutes, preferably less than 150 minutes, or even a duration of 5 to 100 minutes.

NON-CROSSLINKED GLYCOSAMINOGLYCAN HMM

In a particular embodiment, a method according to the invention also considers the implementation of at least one non-crosslinked glycosaminoglycan with a molar mass greater than or equal to 0.5 MDa, preferably greater than 1 MDa, even more preferably comprised of 1 MDa to 4 MDa, called "non-crosslinked HMM glycosaminoglycan".

Preferably, said "non-crosslinked HMM glycosaminoglycan" is implemented in an aqueous solution. This aqueous solution can be distinct from or common to one of the aqueous solutions considered in steps a) and b).

According to a variant embodiment, all or part of said "non-crosslinked HMM glycosaminoglycan" is implemented in the aqueous solution of step b).

According to another variant embodiment, all or part of said “non-crosslinked HMM glycosaminoglycan” is implemented in the form of an aqueous solution distinct from the aqueous solutions of steps a) and b). According to this variant, the aqueous solution containing all or part of said “non-crosslinked HMM glycosaminoglycan” and the aqueous solution from step b) are mixed concomitantly or successively with the aqueous solution from step a).

AQUEOUS GEL

At the end of step c), a homogeneous aqueous gel is obtained, containing at least one "non-crosslinked FMM glycosaminoglycan" and "HMM glycosaminoglycan", including at least one "crosslinked HMM glycosaminoglycan".

Advantageously, an aqueous gel in accordance with the invention may comprise a total concentration of glycosaminoglycans comprised from 10 to 40 mg/g, preferentially from 20 to 30 mg/g relative to its total mass.

An aqueous gel in accordance with the invention can also be characterized by a mass ratio "non-crosslinked FMM glycosaminoglycan" to "HMM glycosaminoglycan" varying from 1:4 to 4:1, preferentially from 1:3 to 5:3, still preferentially from 1:2 to 2:1.

According to a particular embodiment, said aqueous gel has a degree of modification of said glycosaminoglycans of less than 5%, preferably less than 3%, even more preferably less than 1%.

As defined above, it is possible to determine the degree of modification of the gel obtained by methods known to those skilled in the art, in particular by implementing a 1H NMR analysis of said gel.

According to one embodiment of the invention, said aqueous gel is obtained at the end of a process implementing:

• in step a), an aqueous solution comprising at least 5 to 15 mg/g of “crosslinked HMM glycosaminoglycan”;
• in step b), an aqueous solution comprising at least 50 to 60 mg/g of “uncrosslinked FMM glycosaminoglycan” and at least 5 to 15 mg/g of “uncrosslinked HMM glycosaminoglycan” and,

said gel formed has a total glycosaminoglycan concentration of 20 to 30 mg/g relative to its total mass.

As representative of the aqueous gel suitable for the invention, mention may very particularly be made of the aqueous gels comprising:

• at least one cross-linked glycosaminoglycan formed from at least one non-cross-linked glycosaminoglycan with a molar mass greater than or equal to 0.5 MDa, preferably greater than 1 MDa, even more preferably between 1 MDa and 4 MDa, called “cross-linked HMM glycosaminoglycan” ;
• at least one non-crosslinked glycosaminoglycan with a molar mass comprised from 0.04 MDa to 0.3 MDa, preferably from 0.08 MDa to 0.1 MDa, called “non-crosslinked FMM glycosaminoglycan”; and, optionally,
• at least one non-crosslinked glycosaminoglycan with a molar mass greater than or equal to 0.5 MDa, preferably greater than 1 MDa, even more preferably between 1 MDa and 4 MDa, called “non-crosslinked HMM glycosaminoglycan”

and having a total concentration of glycosaminoglycan comprised from 15 to 40 mg/g, and preferably from 20 to 30 mg/g relative to its total mass.

Of course, an aqueous gel in accordance with the invention can also comprise one or more additives conventionally considered in this type of galenics and in particular one or more additives as defined above.

Of course, the choice and the quantity of additive(s) are adjusted so as not to raise any risk of incompatibility with the other compounds used in a gel according to the invention, in particular with said glycosaminoglycans and to be compatible with the uses as described below. These adjustments fall within the general skills of a person skilled in the art.

Thus, in a particular embodiment, the present invention relates to a method as described above further comprising the implementation of at least one additive chosen from the group consisting of anesthetic agents, antioxidants, amino acids, vitamins, minerals, nucleic acids, nucleotides, nucleosides, co-enzymes, adrenergic derivatives, sodium dihydrogen phosphate mono-hydrate and/or di-hydrate and sodium chloride.

In a preferred embodiment, said additive is an anesthetic agent as defined above and in particular lidocaine, lamepivacaine or one of their salts such as the hydrochloride.

The gels obtained according to the process of the invention are very particularly useful as compositions for use in the prevention and/or treatment of an alteration in the surface appearance of the skin.

These compositions can be administered topically but also by injection into the superficial areas of the skin, i.e. into the upper dermis and the middle dermis.

When an injection is considered, it is of course necessary that the aqueous gel be sterile.

Thus, according to one aspect of the present invention, in order to maintain the sterility of said gel, the raw materials used are sterile and the preparation of said gel including its packaging in its administration device is carried out under controlled atmosphere conditions.

According to another aspect, the present invention relates to a process for the preparation of a sterile and injectable composition comprising at least one step consisting in implementing an aqueous gel obtained by a process according to the invention and a step of sterilization in particular, for example in the autoclave.

More specifically, such a method comprises at least one step consisting in implementing an aqueous gel prepared by a method comprising at least the steps consisting in:

1. have an aqueous solution comprising at least one crosslinked glycosaminoglycan formed from non-crosslinked glycosaminoglycan with a molar mass greater than or equal to 0.5 MDa, preferably greater than 1 MDa, even more preferably between 1 MDa and 4 MDa; called “cross-linked HMM glycosaminoglycan”;
2. have an aqueous solution comprising at least one non-crosslinked glycosaminoglycan with a molar mass of 0.04 MDa to 0.3 MDa, preferably 0.08 MDa to 0.1 MDa; called “uncrosslinked FMM glycosaminoglycan”;
3. forming a homogeneous mixture of all or part of said solutions of steps a) and b); And,

a sterilization step in particular, for example in an autoclave

This sterilization is preferably carried out on a composition already packaged in its administration device, generally a syringe.

Preferably, said method comprises a single sterilization step.

Preferably, said sterilization step is carried out by thermal means, for example in an autoclave, in particular at a temperature between 120°C and 140°C.

In particular, the sterilization step is carried out in an autoclave, under moist heat conditions, at a temperature greater than or equal to 121°C to obtain an F0 greater than 15 (sterilizing value).

According to yet another of its aspects, the present invention relates to an injectable composition sterilized according to a sterilization process as described above.

By way of representative of such a composition, mention may in particular be made of those comprising at least

• 0.6 to 0.8% of at least one “HMM cross-linked glycosaminoglycan”;
• 1.4 to 1.8% of at least one “FMM non-crosslinked glycosaminoglycan”; And,
• 0.2 to 0.4% of at least one “HMM non-crosslinked glycosaminoglycan”;

and having a total concentration of glycosaminoglycan between 20 and 30 mg/g, a phase angle (δ) between 25° and 35°, a G' less than or equal to 60 Pa and an extrusion force less than or equal to 15 N when measured with a dynamometer, at a fixed speed of approximately 12.5 mm/min, in syringes of external diameter greater than or equal to 6.3 mm, with a needle of external diameter less than or equal to 0 .3 mm (30 G) and ½'' length, at room temperature.

According to one of its additional aspects, the present invention relates to a kit comprising a pre-filled syringe comprising a sterile and injectable composition as defined above.

According to another aspect, the present invention relates to a sterile and injectable gel or composition according to the invention for its use in the prevention and/or treatment of an alteration in the surface appearance of the skin. The invention relates to a composition according to the invention for its use in the augmentation and/or the filling of soft tissues, in particular the filling of wrinkles.

In particular, the present invention relates to a pre-filled syringe comprising a sterile and injectable composition obtained by the method according to the invention.

The present invention also relates to a kit comprising a pre-filled syringe comprising a sterile and injectable composition obtained by the method according to the invention and instructions for use.

A composition according to the invention is also described for its use in the prevention and/or treatment of an alteration in the viscoelastic or biomechanical properties of the skin.

A composition according to the invention is also described for its use in the prevention and/or treatment of cutaneous signs of chronological aging and/or induced by external factors such as stress, atmospheric pollution, tobacco or prolonged exposure. to ultraviolet (UV).

Materials

Separate sets of samples have been prepared for the different examples below. There are inevitable variations between these series, in particular due to the use of different batches of hyaluronic acid, or even different sterilization cycles. Therefore, it is important to note that comparisons between these series, inter-examples, are not appropriate.

Methods

The following protocols are applied for the study of the viscoelastic properties of the compositions analyzed in the examples.

Characterization of samples in r heology

The viscoelastic properties of a composition are measured by rheometry at room temperature (TA Instruments DHR2) with a cone/plane geometry (cone angle 1°/plate diameter 40 mm). A stress sweep measurement at a frequency of 1 Hz is performed and the elastic modulus (G', in Pa), the phase angle (δ, in °) and the complex viscosity (η*) are measured for a stress of 5 Pa.

Characterization of sample extrusion forces

The extrusion forces of the samples are determined using a dynamometer, at a fixed speed of 12.5 mm/min, a descent path of 25 mm, in 1 mL COC syringes with an internal diameter of 6.4 mm, with a 30 G ½'' needle, at room temperature.

Characterization of sample resistance to degradation over time and heat

The sterilization of injectable hyaluronic acid gels is generally carried out by autoclave (humid heat sterilization process).

The storage stability of these compositions can be evaluated in an accelerated manner, through forced degradation tests at high temperature (ASTM F1980-16).

Gel degradation is characterized by a significant loss of elastic modulus (G').

In this context, the percentage loss of G' on sterilization (ΔG' (%)) constitutes a relevant indicator of the resistance of the gel to degradation, both over time and to heat sterilization.

For this, the variation of G' on sterilization is calculated as follows:

ΔG' (%) = [(G' after sterilization – G' before sterilization) / G' before sterilization] x 100

In addition, a percentage improvement in G' can be defined as follows:

Improvement G'(%) = [(ΔG' reference sample - ΔG' study sample)/ ΔG' reference sample] x 100

Characterization of sample resistance to enzymatic degradation

To study the resistance of the samples to enzymatic degradation, 2g of the gel tested are brought into contact and homogenized with a hyaluronidase (HAase) in solution in saline phosphate buffer (0.5 U/mL of gel), then placed in an oven at 37 °C for 24 hours.

Each sample is characterized by a rheology measurement before and after the enzymatic treatment and the variation of G' before/after degradation is calculated as follows: ΔG' = [(G' final – G' initial) / G' initial] x 100 This variation of G' is a direct index of the degree of degradation of the gel in the presence of HAase.

Example 1

Effect of supplementation with a composition based on HMM cross-linked hyaluronic acid with an FMM hyaluronic acid

The samples studied are shown below.

Comparative sample 1-1 is an autoclaved syringe-sterilized cross-linked hyaluronic acid gel prepared from HMM hyaluronic acid (1.5 MDa, HTL, Ph. Eur. pharmaceutical/medical grade) and BDDE (Sigma Aldrich, ≥ 95%) with a total hyaluronic acid concentration of 20mg/g and a degree of modification of 2%.

Sample 1-2, according to the invention, is a cross-linked hyaluronic acid gel sterilized in an autoclave syringe, with a total hyaluronic acid concentration of 20 mg/g and prepared from a 1:1 mixture of sample 1-1 before sterilization with a 20 mg/g solution of non-crosslinked FMM sodium hyaluronate (0.1 MDa, HTL, Ph. Eur. pharmaceutical/medical grade).

Comparative sample 1-3 is a cross-linked hyaluronic acid gel sterilized in an autoclave syringe, with a total hyaluronic acid concentration of 10mg/g and prepared from a 1:1 mixture of sample 1- 1 before sterilization with a non-crosslinked hyaluronic acid-free phosphate-buffered saline solution.

The characteristics of the samples, their G' and their loss of G' on sterilization are summarized in Table 1 below and in Figure 1.

Samples HMM cross-linked hyaluronic acid concentration (mg/g) Concentration of non-crosslinked hyaluronic acid FMM ( 0.1 MDa) (mg/g) Total hyaluronic acid concentration (mg/g) ΔG’ (%) 1-1 (reference) 20 0 20 -10.8 1-2 (invention) 10 10 20 -2.4 1-3 (comparative) 10 0 10 -21.6

The results show better resistance to degradation for compositions comprising an FMM hyaluronic acid (sample 1-2 according to the invention) compared to a composition with the same total concentration of hyaluronic acid but not comprising any FMM hyaluronic acid ( sample 1-1 (reference)).

Furthermore, it emerges that the higher the concentration of cross-linked HMM hyaluronic acid, the greater the stability of the composition. Thus, sample 1-3 (comparative, 10 mg/g of HMM hyaluronic acid) presents a greater loss of G' (ΔG'= -21.6%) than sample 1-1 (comparative) comprising 20 mg/g of HMM hyaluronic acid (ΔG'=-10.8%). Nevertheless, this increase in stability is much lower than that observed for sample 1-2 (invention) comprising non-crosslinked hyaluronic acid FMM (ΔG'= -2.4%).

Consequently, it is not the simple increase in the total concentration of hyaluronic acid which is associated with the increase in the resistance of the compositions to degradation but rather the supplementation of the composition based on HMM hyaluronic acid concerned.

Example 2

Effect of supplementation with a composition based on HMM cross-linked hyaluronic acid , optionally comprising a non-crosslinked HMM hyaluronic acid, with an FMM hyaluronic acid

A 2-1 sample is prepared as follows.

Preparation of a BDDE-crosslinked hyaluronic acid gel in a basic medium from non-crosslinked HMM hyaluronic acid (HTL, 1.5 MDa, pharmaceutical/medical grade Ph. Eur.). The crosslinked fraction obtained is neutralized and swollen in saline phosphate buffer so as to obtain a final gel at 15 mg/g in NaHA. He is then on dialysis. At this stage, the gel has an elastic modulus G' of approximately 80 Pa to 120 Pa and a phase angle δ of 7° to 10°.

A 15 mg/g solution of non-crosslinked HMM sodium hyaluronate (4 MDa, HTL, pharmaceutical/medical grade Ph. Eur.) is added to the crosslinked fraction to reach a proportion of 30% by mass relative to the total mass of hyaluronic acid in the composition. The gel is homogenized, extruded through a grid and filled into 1mL COC syringes. Finally, the gel is sterilized in an autoclave .

Sample 2-2 is prepared similarly to sample 2-1 but the composition of the non-crosslinked hyaluronic acid gel used has been modified so that the final gel also includes 10 mg/g of FMM hyaluronic acid non-crosslinked (0.1 MDa, HTL, Ph. Eur. pharmaceutical/medical grade).

Sample 2-3 is prepared like sample 2-1 but without non-crosslinked 4 MDa hyaluronic acid.

Sample 2-4 is prepared like sample 2-1 but replacing the non-crosslinked 4 MDa hyaluronic acid used with 0.1 MDa hyaluronic acid.

For each of these samples, the variation (in percentage) of G' is calculated. The results obtained are summarized in the tables below.

Table 2 presents the stabilization of a mixture of cross-linked and non-cross-linked hyaluronic acid.

Samples HMM hyaluronic acid Added non-crosslinked FMM hyaluronic acid ( 0.1 Mda) ΔG’ (%) 2-1 (comparison) reticulated + unreticulated No -25 2-2 (invention) reticulated + unreticulated Yes -15

Table 3 presents the stabilization of cross-linked hyaluronic acid alone.

Samples HMM hyaluronic acid Added non-crosslinked FMM hyaluronic acid ( 0.1 Mda) ΔG’ (%) 2-3 (comparative) reticular alone No -15 2-4 (invention) reticular alone Yes -10

The results show that the protective effect of non-crosslinked FMM hyaluronic acid during autoclave sterilization is observed both for compositions comprising cross-linked HMM hyaluronic acid alone (sample 2-4 vs. sample 2-3) and for compositions comprising a mixture of cross-linked and non-cross-linked HMM hyaluronic acid (sample 2-2 vs. sample 2-1).

Thus, the addition of non-crosslinked FMM hyaluronic acid to compositions comprising a cross-linked HMM hyaluronic acid alone (sample 2-4) or in a mixture with a non-cross-linked HMM hyaluronic acid (sample 2-2) advantageously makes it possible to increase their resistance to degradation.

Example 3

Study of rheology And of the stability hyaluronic acid gels including of the' hyaluronic acid No reticular of different e s molar masses

Rheology of hyaluronic acid solutions of different molar mass

Solutions of 10 mg/g (1%) of non-crosslinked hyaluronic acid of variable molar masses (0.04 MDa, 0.1 MDa, 0.2 MDa and 1.5 MDa, HTL, pharmaceutical/medical grade Ph. Eur.) are prepared in phosphate buffered saline. The elastic modulus (G') and the complex viscosity (η*) of each of these fractions are determined and detailed in Table 4 below. [Table 4] Samples Non-crosslinked hyaluronic acid (MDa) η* (mPa.s) before sterilization G' (Pa) before sterilization 3-1 0.04 n/a** n/a** 3-2 0.1 n/a** n/a** 3-3 0.2 21.2 <1 3-4 1.5 2110 6.0

**ND Not Determinable at a stress of 5Pa ( sample 3-1: η* = 2.55 mPa.s and G' = 0.65 Pa at 0.6Pa; sample 3-2: η* = 5.2 mPa. s and G' = 0.0057 Pa at 1Pa ).

The results above show that a gel based on non-crosslinked hyaluronic acid with a molar mass less than or equal to 0.2 MDa alone and concentrated at 1% does not exhibit any significant elastic properties and a very low viscosity.

Mechanical contribution

A mixture of cross-linked and non-cross-linked HMM hyaluronic acid, sample 3-5 (reference), is prepared as follows. Preparation of a BDDE-crosslinked hyaluronic acid gel in a basic medium from non-crosslinked HMM hyaluronic acid (1.5 MDa, HTL, Ph. Eur. pharmaceutical/medical grade). The crosslinked fraction obtained is neutralized and swollen in saline phosphate buffer so as to obtain a final gel at 15 mg/g in NaHA. He is then on dialysis. At this stage, the gel has an elastic modulus G' of approximately 80 to 120 Pa and a phase angle δ of 7 to 10°. A 15 mg/g solution of non-crosslinked HMM sodium hyaluronate (4 MDa, pharmaceutical/medical grade Ph. Eur.) is added to the crosslinked fraction to reach a proportion of 30% by mass relative to the total mass of hyaluronic acid in the composition. The gel is homogenized, extruded through a grid and filled into 1 mL COC syringes. Finally, the gel is sterilized in an autoclave .

Samples 3-6 to 3-9 are prepared similarly to sample 3-5 but with a non-crosslinked hyaluronic acid gel further comprising an additional fixed concentration of 10 mg/g of non-crosslinked hyaluronic acid relative to to the total mass of the composition (1%).

For this addition, non-crosslinked hyaluronic acids of different molar masses are used, namely for samples 3-6 to 3-9, in order: 0.04 MDa, 0.1 MDa, 0.2 MDa and 1 .5 MDa, HTL, pharmaceutical/medical grade Ph. Eur.).

The rheological characteristics of these samples are determined before and after sterilization, on 6 syringes of each sample. The results after sterilization are summarized in Table 5 below and in Figure 2.

Samples Added non-crosslinked hyaluronic acid (MDa) δ (°)/ standard deviation (°) G’ (Pa) before sterilization / deviation you type (Pa) G’ (Pa) after sterilization / standard deviation (Pa) F (N) / standard deviation (N) 3-5 (reference) - 16.4 / 0.5 137 / 3 110 / 3 10.4 / 0.6 3-6 (invention) 0.04 16.8 / 0.4 138 / 2 111 / 3 12.0 / 0.3 3-7 (invention) 0.1 17.2 / 0.4 144 / 5 124 / 5* 14.8 / 0.5 3-8 (invention) 0.2 19.9 / 0.7 161 / 3 135 / 9* 15.6 / 0.4 3-9 (comparative) 1.5 27.6 / 4 305 / 3 240 / 21* 16.0 / 0.7

*Mean significantly different from Reference sample mean, p < 0.001, two-sample t-test.

Thanks to a limited mechanical contribution of the non-crosslinked FMM hyaluronic acid, samples 3-6 to 3-8 remain flexible gels, i.e. gels with a δ between 15° and 45°, a G' less than or equal to 150 Pa and an extrusion force less than or equal to 18N.

It is noted that the presence of an additional FMM hyaluronic acid and therefore of an increased concentration of hyaluronic acid in the gels according to the invention does not affect their flexibility.

On the other hand, the comparative sample 3-9, comprising an additional hyaluronic acid HMM, presents very significantly increased rheological characteristics and can no longer be considered as a flexible gel (G' > 150 Pa).

Resistance to degradation

The resistance to heat degradation of each sample is studied by calculating the percentage of G' lost after undergoing the same cycle of sterilization in the autoclave l.

The results obtained are summarized in Table 6 below, in Figure 2 and Figure 3.

Samples Added non-crosslinked hyaluronic acid (MDa) ΔG’ (%) Improvement G' (%) 3-5 (reference) - -20 0 3-6 (invention) 0.04 -19 2 3-7 (invention) 0.1 -13 32 3-8 (invention) 0.2 -16 20 3-9 (comparative) 1.5 -21 -8

As shown in Figure 2, under the sterilization conditions implemented (identical for all the samples tested), the reference sample 3-5 undergoes a loss of around 20%.

The addition of a non-crosslinked HMM hyaluronic acid (1.5MDa, sample 3-9) does not improve the loss of G' on sterilization, which remains higher than that of the reference sample 3-5 ( 20%).

On the contrary, the supplementation according to the invention, that is to say the addition of a hyaluronic acid of molar mass comprised from 0.04 MDa to 0.2 MDa, more preferably from 0.08 MDa to 0.1 MDa , is at the origin of a decrease in the loss of G' on sterilization (comparison of samples 3-6 to 3-8 with sample 3-5), in other words, at the origin of an increase the resistance to degradation of the composition.

A composition supplemented with an FMM hyaluronic acid according to the invention therefore has better resistance to degradation in comparison with compositions having an identical total concentration of hyaluronic acid, the same rate of crosslinking of the fraction of crosslinked HMM hyaluronic acid but no supplementation.

This improvement is minimal but already noticeable from the lowest molar mass (0.04 MDa) of hyaluronic acid used (sample 3-6). This improvement is significant with hyaluronic acid of 0.1 MDa and 0.2 MDa (samples 3-7 and 3-8).

Thus, the comparison of the means from the 6 measurements on 6 syringes of each product after sterilization makes it possible to demonstrate that samples 3-7 and 3-8 have a value of G' significantly higher than that of sample 3-5 (Figures 2 and 3).

This increase in G' is linked both to the interactions between the FMM hyaluronic acid and the HMM hyaluronic acid used in the formulation and especially to the effect of improving resistance to degradation associated with the presence of hyaluronic acid. FMM in the composition (visible by with the improvement of the values of loss of G' on sterilization).

Thus, while the addition of a non-crosslinked HMM hyaluronic acid (for example 1.5 MDa) increases the mechanical properties of the composition without significantly improving its resistance to degradation, the supplementation with a non-crosslinked FMM hyaluronic acid allows to increase the resistance to degradation of a gel while retaining the mechanical properties of a flexible gel.

Example 4

Influence of variation in supplementation

For this example, comparative sample 4-1 is identical to sample 3-5 (comparative) prepared in Example 3. Different samples were prepared in order to study the effect of different concentrations of hyaluronic acid FMM not cross-linked.

Sample 4-1 is compared to samples 4-2 to 4-4 prepared in a similar way but for which the composition of the uncrosslinked hyaluronic acid gel used also comprises increasing concentrations of uncrosslinked FMM hyaluronic acid (0 .1 MDa HTL, pharmaceutical/medical grade Ph. Eur.).

The proportion of non-crosslinked FMM hyaluronic acid (0.1 MDa, HTL, pharmaceutical/medical grade Ph. Eur.) corresponds to the ratio between the mass of non-crosslinked FMM hyaluronic acid and the mass of cross-linked HMM hyaluronic acid in the composition (%).

The rheological characteristics of the samples and their loss of G' on sterilization ΔG' (%) are summarized in Table 7 below and in Figure 4.

Samples Proportion of non-crosslinked hyaluronic acid FMM (%) Concentration of uncrosslinked FMM hyaluronic acid (mg/g) Hyaluronic acid concentration Cross-linked and non-cross-linked HMM (mg/g) δ (°) after sterilization G’ (Pa) after sterilization ΔG’ (%) F(N) 4-1 (comparison) 0 0 15 16.4 / 0.5 110 / 3 -20 10.4 / 0.6 4-2 (invention) 48 5 15 16.7 / 0.4 119 / 2 -15 12.4 / 0.5 4-3 (invention) 95 10 15 17.2 / 0.4 124 / 5 -13 14.8 / 0.5 4-4 (invention) 143 15 15 18.1 / 0.1 120 / 6 -16 17.4 / 0.4

The stabilizing effect of supplementation is visible on all of the samples 4-2 to 4-4 in accordance with the invention.

It is therefore possible to obtain the advantageous effect of the supplementation of gels comprising at least one cross-linked HMM hyaluronic acid with more or less significant amounts of non-cross-linked FMM hyaluronic acid.

In addition, the results above show the obtaining of compositions highly concentrated in hyaluronic acid (up to 30 mg/g in total hyaluronic acid) which remain flexible and easily injectable whether it is during the implementation of low proportions in non-crosslinked FMM hyaluronic acid (about 50%) or higher proportions of non-crosslinked FMM hyaluronic acid (about 150%)).

Example 5

Influence of v change in hyaluronic acid concentration HMM

For this example, comparative sample 5-1 is prepared in the same way as sample 3-5 (comparative) prepared in Example 3. Samples 5-2 and 5-3 in accordance with the invention and comprising a concentration of HMM hyaluronic acid lower than that of the preceding examples were prepared with the aim of studying the effect of a fixed concentration of non-crosslinked FMM hyaluronic acid on compositions of different concentrations of cross-linked and non-cross-linked HMM hyaluronic acids.

Sample 5-2 is prepared in a similar manner to sample 5-1 but with a target concentration of 12 mg/g for the cross-linked hyaluronic acid fraction and 12 mg/g for the non-HMM hyaluronic acid solution. cross-linked to which is added a sufficient quantity of non-cross-linked hyaluronic acid 0.1 MDa (HTL, pharmaceutical/medical grade Ph. Eur.) to achieve a concentration of 16 mg/g of non-cross-linked hyaluronic acid 0.1 MDa in the final composition.

Sample 5-3 is also prepared similarly to the Reference sample but with a target concentration of 9 mg/g for the cross-linked HMM hyaluronic acid fraction and 9 mg/g for the HMM hyaluronic acid solution non-crosslinked to which is added a sufficient quantity of non-crosslinked FMM hyaluronic acid 0.1 MDa (HTL, pharmaceutical/medical grade Ph. Eur.) to achieve a concentration of 16 mg/g of non-crosslinked hyaluronic acid 0.1 MDa in the final composition.

The rheological characteristics of samples 5-1 to 5-3, their proportion of non-crosslinked hyaluronic acid FMM (%) and their loss of G' on sterilization (ΔG' (%)) are summarized in Table 8 below.

Samples Proportion of non-crosslinked hyaluronic acid FMM (%) Concentration of cross-linked and non-cross-linked hyaluronic acid HMM (mg/g) FMM uncrosslinked hyaluronic acid concentration (mg/g) G’ (Pa) after sterilization δ (°) after sterilization ΔG’ (%) F(N) after sterilization 5-1 (comparison) 0 15 0 98±4 17.7±0.5 -25 11.9±0.3 5-2 (invention) 190 12 16 74±1 19.1 ± 0.6 -19 18.2±0.3 5-3 (invention) 253 9 16 47±8 21.3 ± 0.8 -4 14.1±0.9

Thanks to the addition of non-crosslinked hyaluronic acid FMM, it is possible to reduce the fraction of cross-linked and non-crosslinked hyaluronic acid HMM from 15 mg/g to 12 mg/g and to observe only a slight decrease in G' (sample 5-1 (comparative) vs. sample 5-2 (invention)).

In addition, thanks to the addition of non-crosslinked FMM hyaluronic acid, it is possible to switch from a 15 mg/g hyaluronic acid gel to a 28 mg/g hyaluronic acid gel while maintaining the mechanical properties. of a flexible gel (sample 5-1 (comparative) vs. sample 5-2 (invention)).

Sample 5-3 prepared with a very small amount of cross-linked and non-cross-linked HMM hyaluronic acid shows a minimal loss of G' on sterilization (only - 4%).

The presence of non-crosslinked FMM hyaluronic acid advantageously makes it possible to reduce the quantity of crosslinked hyaluronic acid necessary to prepare a flexible gel. It also increases the total concentration of hyaluronic acid. In other words, supplementation therefore makes it possible to obtain flexible gels with potentially increased biocompatibility (reduction in the proportion of cross-linked hyaluronic acid used) but also effective from a biomechanical and/or biological point of view.

Example 6

Resistance to enzymatic degradation

Sample 6-1 is prepared according to the same protocol as sample 3-5 (comparative) prepared in Example 3.

Sample 6-2 is prepared in a similar manner but the composition of the non-crosslinked hyaluronic acid gel used has been modified so that the final gel also comprises 10 mg/g of non-crosslinked FMM hyaluronic acid (0.1 MDa , HTL, pharmaceutical/medical grade Ph. Eur.).

Sample 6-3 is also prepared in a similar way to sample 6-1 but the composition of the uncrosslinked hyaluronic acid gel used has been modified so that the final gel also includes 10 mg/g of hyaluronic acid Uncrosslinked HMM (1.5 MDa, HTL, Ph. Eur. pharmaceutical/medical grade).

All of these samples are subjected to an enzymatic degradation test using hyaluronidase (HAase).

The rheological characteristics of the samples and their loss of G' to enzymatic degradation are summarized in Table 9 below and in Figure 5.

Samples Concentration of cross-linked and non-cross-linked hyaluronic acid HMM (mg/g) Concentration of uncrosslinked hyaluronic acid added (mg/g) Added non-crosslinked hyaluronic acid (MDa) G’ (Pa) before degradation / standard deviation G’ (Pa) after degradation / standard deviation ΔG’ (%) 6-1 (comparison) 15 0 N / A 110 / 3 89 / 1 -19 6-2 (invention) 15 10 0.1 124 / 5 110 / 3 -12 6-3 (comparative) 15 10 1.5 240 / 21 188 / 4 -22

It is noted that samples 6-2 and 6-3 (comprising respectively hyaluronic acids with molar masses of 0.1 MDa and 1.5 MDa) have very different starting properties. The properties of sample 6-3 are not desirable for cutaneous applications for regeneration and/or improvement of the appearance of the skin (see δ, G' and F).

The loss of G' to enzymatic degradation is lower in the presence of a non-crosslinked FMM hyaluronic acid than without (sample 6-2 vs. sample 6-1) and even than in the presence of non-crosslinked HMM hyaluronic acid ( sample 6-2 vs. sample 6-3). The increased resistance of the composition presented as in accordance with the invention (sample 6-2) is therefore not associated with the simple addition of non-crosslinked hyaluronic acid but quite specifically with the addition of non-crosslinked FMM hyaluronic acid.

The compositions supplemented according to the invention therefore have better resistance to enzymatic degradation in comparison with compositions having the same total concentration of hyaluronic acid, the same degree of crosslinking of the fraction of crosslinked hyaluronic acid but comprising only hyaluronic acid hyaluronic HMM.

This increased resistance to degradation by hyaluronidase predicts the increased in vivo durability of the compositions according to the invention.

Finally, the above results highlight the protective effect of non-crosslinked FMM hyaluronic acid, and therefore the beneficial effect of supplementation with a composition based on at least one crosslinked glycosaminoglycan.

Claims (26)

Process for the preparation of an aqueous gel comprising at least one cross-linked glycosaminoglycan and at least one non-cross-linked glycosaminoglycan, said process comprising at least the steps consisting in:

1. have an aqueous solution comprising at least one cross-linked glycosaminoglycan, called “cross-linked HMM glycosaminoglycan”, formed from non-cross-linked glycosaminoglycan with a molar mass greater than or equal to 0.5 MDa, preferably greater than 1 MDa, even more preferably comprised of 1 MDa to 4 MDa and a crosslinking agent;
2. have an aqueous solution comprising at least one non-crosslinked glycosaminoglycan with a molar mass of from 0.04 MDa to 0.3 MDa, preferably from 0.08 MDa to 0.1 MDa; called “uncrosslinked FMM glycosaminoglycan” and,
3. form a homogeneous mixture of all or part of said solutions of steps a) and b).

Process according to Claim 1, characterized in that the said "crosslinked HMM glycosaminoglycan" is prepared with a ratio between the mass of crosslinking agent and the mass of uncrosslinked glycosaminoglycan used for the preparation of the said crosslinked glycosaminoglycan of less than or equal to 8% by mass, preferably less than or equal to 5%, and even more preferably less than 2%. Process according to Claim 1 or 2, characterized in that the said crosslinking agent used for the formation of the “crosslinked HMM glycosaminoglycan” is a bi- or multifunctional epoxy crosslinking agent, preferably 1,4-butanediol diglycidyl ether. Process according to one of Claims 1 to 3, further comprising the use of at least one non-crosslinked glycosaminoglycan with a molar mass greater than or equal to 0.5 MDa, preferentially greater than 1 MDa, even more preferentially comprised from 1 MDa to 4 MDa, known as “non-crosslinked HMM glycosaminoglycan”. Process according to Claim 4, characterized in that all or part of the said “non-crosslinked HMM glycosaminoglycan” is implemented in the aqueous solution of stage b). Process according to Claim 4, characterized in that all or part of the said “non-crosslinked HMM glycosaminoglycan” is implemented in the form of an aqueous solution distinct from the aqueous solutions of stages a) and b). Process according to Claim 6, characterized in that in step c) the aqueous solution of said "non-crosslinked HMM glycosaminoglycan" and the aqueous solution of step b) are mixed concomitantly or successively with the aqueous solution of step a ). Process according to any one of Claims 1 to 7, characterized in that stage c) comprises at least one operation of extruding the mixture formed through at least one grid. Process according to any one of Claims 1 to 8, characterized in that it comprises the use of at least 5 mg/g of “crosslinked HMM glycosaminoglycan” relative to the total mass of the said aqueous gel. Process according to any one of Claims 1 to 9, characterized in that it comprises the use of at least 3 mg/g, preferably 10 mg/g, of the said "non-crosslinked FMM glycosaminoglycan" with respect to the mass total of said aqueous gel. Process according to any one of Claims 1 to 10, characterized in that the said aqueous gel has a total concentration of glycosaminoglycans comprised from 10 to 40 mg/g, preferentially from 20 to 30 mg/g relative to the total mass of the said aqueous gel. . Process according to any one of Claims 1 to 11, characterized in that it comprises the use of glycosaminoglycans in a “non-crosslinked FMM glycosaminoglycan” mass ratio to “HMM glycosaminoglycan” of 1:4 to 4:1, preferably of 1:3 to 5:3, more preferably 1:2 to 2:1. Process according to any one of Claims 1 to 12, characterized in that the said aqueous gel has a degree of modification of the said glycosaminoglycans of less than or equal to 5%, preferably less than or equal to 3%, even more preferably less than or equal to 1%. Process according to any one of Claims 2 to 13, characterized in that it implements:

• in step a), an aqueous solution comprising at least 5 to 15 mg/g of “crosslinked HMM glycosaminoglycan”;
• in step b), an aqueous solution comprising at least 50 to 60 mg/g of "uncrosslinked FMM glycosaminoglycan" and at least 5 to 15 mg/g of "uncrosslinked HMM glycosaminoglycan" and in that,

said gel formed has a total glycosaminoglycan concentration of 20 to 30 mg/g relative to its total mass. Process according to any one of Claims 1 to 14, characterized in that the said glycosaminoglycans are chosen from hyaluronic acid, heparosan, chondroitin sulphate, dermatan sulphate, keratan sulphate and their mixtures, and preferentially are 'hyaluronic acid. Process according to any one of Claims 1 to 4, characterized in that the said chosen glycosaminoglycans are of different or identical, preferably identical, chemical nature. Process according to any one of Claims 1 to 16, characterized in that it also comprises the use of at least one additive chosen from the group consisting of anesthetic agents, antioxidants, amino acids, vitamins , minerals, nucleic acids, nucleotides, nucleosides, co-enzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and/or dihydrate and sodium chloride. Aqueous gel of glycosaminoglycans obtainable by a process according to any one of claims 1 to 17. Process for the preparation of a composition, sterile and injectable, characterized in that it comprises at least one step of implementing an aqueous gel obtained according to one of Claims 1 to 17 or as defined in Claim 18 ; and, a sterilization step, for example in an autoclave. Process according to Claim 19, characterized in that the said composition is packaged in a syringe prior to its sterilization. Composition, sterile and injectable, obtained by the process according to any one of claims 19 or 20. Composition according to Claim 21 for its use in the prevention and/or treatment of an alteration in the surface appearance of the skin. Aqueous gel according to Claim 18 for its use in the prevention and/or treatment of an alteration in the surface appearance of the skin Composition according to Claim 21 for its use in augmenting and/or filling soft tissues, in particular filling wrinkles. A pre-filled syringe comprising a composition according to claim 21. Kit comprising a pre-filled syringe according to claim 25 and instructions for use.